PROJECT SUMMARY The central objective of this research is to comprehensively investigate the variation, regulation, and functional consequences of RNA editing in human transcriptomes. RNA editing has emerged as an important and widespread mechanism for generating transcriptome diversity in eukaryotic cells. Aberrant RNA editing has been implicated in a variety of diseases including neurological diseases and cancer. The most abundant type of RNA editing is the A-to-I RNA editing (the deamination of adenosine to inosine) mediated by the ADAR family of RNA editing enzymes. High-throughput RNA sequencing studies have revealed millions of A-to-I RNA editing sites in the human transcriptome. Despite the explosion in the number of identified RNA editing sites, there remain significant knowledge gaps about the regulation and function of RNA editing. The landscapes of RNA editing can be dynamically regulated among different tissues or cell types or in response to stimuli, as well as become dysregulated in diseases. While previous studies on the regulation of RNA editing mainly focused on the ADAR enzymes, the roles of other trans-acting regulators such as RNA binding proteins have largely been unexplored. Moreover, the functions of most A-to-I RNA editing events are currently unknown. Previous functional studies have investigated RNA editing events in coding regions of selected genes. Recent data indicate that the vast majority of A-to-I RNA editing events occur in non-coding regions, such as 5'-UTR, intron, and 3'-UTR, suggesting widespread regulatory effects of editing on the RNA. Indeed, RNA editing may influence a variety of regulatory processes at the post-transcriptional level, such as the regulation of RNA splicing, localization, stability, and translational efficiency. Of note, work from us and others has shown that RNA editing can create or disrupt functional microRNA target sites in the 3'-UTR, suggesting that RNA editing can directly regulate mRNA translation or stability. In three complementary and tightly integrated research aims, we will investigate the regulation of RNA editing by trans-acting regulators and environmental stimuli (Aim 1), the genetic variation and phenotypic association of RNA editing in human populations (Aim 2), and the functional consequences of RNA editing on mRNA translation and stability (Aim 3). Collectively, the proposed studies will provide significant insights into the regulation, genetic variation, and function of RNA editing. Additionally, through this project we will develop innovative approaches for quantitative analyses of RNA editing using a variety of transcriptome sequencing technologies. We anticipate that these approaches will be broadly useful for studying RNA editing as well as other types of RNA variants and modifications in eukaryotic transcriptomes.